Overall aim of this grant application is to elucidate the causal relationship between endothelial dysfunction, oxidative stress, and the inflammatory cytokine, tumor necrosis factor alpha (TNF-(). Accordingly, this proposal will test the hypothesis that inflammation produces endothelial dysfunction in Type II diabetes, which is a leading risk factor for coronary heart disease. There are three aims, and associated with each of the aims are three or four hypotheses. The first aim will determine if TNF-( plays a critical role in diabetic endothelial dysfunction. We propose that: 1). In dbTNF-/dbTNF- mice endothelial dilation will be greater than that in diabetic (db/db) mice, and comparable to Db/db controls. Decrements in endothelial function in db/db mice will progressively increase from 6 to 12 to 18 weeks. 2). Administration of neutralizing antibodies to TNF-( will ameliorate endothelial dysfunction in db/db mice of varying ages, 6, 12 and 18 weeks. The effect will be most prominent in the older animals. 3). Expression of TNF-( and TNF-( receptors (TNFRs: TNFR1 and TNFR2) are elevated in coronary arterioles, even at the age of 6 weeks before the development of diabetes, in db/db mice compared to Db/db mice. We propose that the expression of TNF-( and its receptors increase progressively with age, and with the development of diabetes. The second aim will determine if TNF-( produces endothelial dysfunction by stimulation of the production of reactive oxygen species (ROS) in db/db mice through activation of NADPH oxidase. We propose to test anti-TNF-( attenuates ROS production in diabetes in db/db mice. The third aim will determine if AGE/RAGE and TNF-( signaling interact to amplify the oxidative stress and induce endothelial dysfunction in diabetic mice. We propose to test if: 1). In dbTNF-/dbTNF- mice, RAGE expression and AGE formation are less than in db/db mice. 2). Activation of the transcription factor NFkB is less in dbTNF-/dbTNF- or db/db + sRAGE mice compared to db/db mice. 3). In db/db mice of varying ages: 6, 12 and 18 weeks, sRAGE restores endothelial dependent dilation toward that of Db/db controls by reducing TNF-( expression and ROS production. 4). Activation of RAGE compromises endothelial dilation to a greater extent in db/db than in Db/db or dbTNF-/dbTNF- mice. 5). In db/db mice, sRAGE reduces ROS production and TNF-( expression compared to that in db/db mice, and this effect will be mediated through decreased expression of NAD(P)H oxidases. We utilize a combination of approaches involving diameter measurements of isolated coronary arterioles from mouse heart, the oxidative fluorescent dye dihydroethidium (DHE-fluorescence imaging) and electron paramagnetic resonance (EPR) to evaluate O2.- production, electrochemical detection of authentic NO, real time PCR of RNA transcripts, and Western Blotting to evaluate expression of key proteins in Db/db control, dbTNF-/dbTNF-, db/db + sRAGE and db/db mice. We believe that this study will provide a solution to the specific problem and new approaches for the longer term; it might represent exciting new targets for the development of drugs for cardiovascular disorders in ischemic heart disease, obesity, diabetes and hypertension.